The invention relates to the fields of detection and amplification of nucleic acids.
The diagnostic use of polymerase chain reaction (PCR) in clinical microbiology has vastly increased our understanding of infection by Chlamydia bacteria (Boman, J. et al. J. Clin. Microbiol. 1999, 37:3791-3799; Johnson, R. et al., J. Clin. Microbiol. 2000, 38:4382-4386). However, several factors impede optimal diagnostic use of the high sensitivity of this method. In particular, all sensitive and specific assays for application to diagnostic microbiology require post-PCR processing by highly skilled personnel. This requirement is particularly true of nested PCR methods that are exceedingly sensitive to cross-contamination by product carryover. In addition, the preservation of nucleic acid targets in specimens is often poor, and the methods of DNA or nucleic acid extraction are inadequate for retrieval of target DNA at low concentration (Apfalter, P. et al. J. Clin. Microbiol. 2001, 39:519-524). Finally, specimens with low amounts of microorganisms, e.g., chlamydiae, have a Poisson distributed sampling variability, so that some aliquots from such specimens result in positive amplification while others remain negative (Smieja, M. et al. J. Clin. Microbiol 2001, 39:1796-1801).
The amount of original specimen analyzed by a single PCR may frequently be the sensitivity-limiting factor. One method for overcoming this limitation is amplifying numerous aliquots of the DNA sample (Smieja, M. et al. J. Clin. Microbiol 2001, 39:1796-1801); however, use of a single PCR reaction is desirable. Quantitative fluorescent real-time PCR methods hold the potential to detect low amounts of DNA in a single sample but have not yet been combined with nucleic acid isolation to result in high-sensitivity, high-throughput platforms suitable for wide use in clinical microbiology. Thus, there is a need for methods for highly sensitive detection of DNA.